View source for Ob/ob Genotyping

Used method described in Ellet et al (http://dx.doi.org/10.1038/oby.2008.443)

==Primers==
* ob/ob RFLP-F: 5'- TGA GTT TGT CCA AGA TGG ACC -3'
* ob/ob RFLP-R: 5'- GCC ATC CAG GCT CTC TGG -3'
* ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3'
* ob/ob - Lepob-R: 5'- GCA GAT GGA GGA GGT CTC A -3'

===Primer Mixes===
Primers are resuspended at 100 uM, so add 3x10 uL of the primers to 970uL of water to make the primer mixes:
*For '''WT Primers''' combine both RFLP primers with the WT-Lep-F primer
*For '''Ob Primers''' combine both RFLP primers with the Lebob-R primer

==PCR Reaction==
The PCR program is called '''ob-ob''' and is:

* 95 °C 2 min 
* 40 cycles of:
** 95 °C 30 s
** 58 °C 30 s
** 72 °C 45 s
*72 °C 7 min

==Bands==
There should be a control band in all lanes at 191bp
For the wt primers if there is one or more wt alleles there should be a band at 104bp
For the ob primers, if there is one or more ob alleles there should be a band at 123bp


[[ Category:Genotyping ]]
[[ Category:Mouse Work]]
[[ Category:PCR]]
[[ Category:DNA]]
[[ Category:Molecular Biology]]